Exploring albumin-sulfonephthalein dyes interactions by a chemometric-assisted and multi-technique equilibria analysis in solution.

Albumin is undoubtedly the most studied protein thanks to its widespread diffusion and biochemistry; despite its binding ability towards different dyes, provoking dye's colour change, has been exploited for decades for quantification purposes, the joint effect of working pH, ionic strength, and dye's pKa still remains only sporadically discussed. In the present study, the interaction of Bovine Serum Albumin (BSA) with five dyes belonging to the sulfonephthalein group, Bromophenol Blue (BPB, pKa = 3.75), Bromocresol Green (BCG, pKa = 4.42), Chlorophenol Red (CPR, pKa = 5.74), Bromocresol Purple (BCP, pKa = 6.05) and Bromothymol Blue (BTB, pKa = 6.72), is investigated at four working pH values (3.5, 6.0, 7.5 and 9.0) and two ionic strength conditions by UV-Vis spectroscopy. Principal Component Analysis is then applied to rationalize dye behavior upon BSA addition at each pH value and to summarize the protein effect on dyes' spectral features, identifying three general behaviors. The most relevant systems are then submitted to further characterization involving a solution equilibria study aimed at determining conditional binding constants for the selected DSA-dye adducts and fluorescence, CD, and 1H NMR spectroscopy to evaluate the binding effect on the species involved.

Dye-decolorization of a newly isolated strain Bacillus amyloliquefaciens W36.

Dye-decolorization is one of the most important steps in dye-polluted wastewater treatment. The dye-decolorization bacteria were isolated from active sludge collected from wastewater treating pond of a dyeing and printing plant using serial dilution method. Among the 44 bacteria isolates from the active sludge, the strain Bacillus amyloliquefaciens W36 was found to have strong ability in dye-decolorization. The effects of carbon source, nitrogen sources, C/N, metal ions, temperature, pH, and rotation speed for dye-decolorization were investigated. The optimum decolorization conditions were that the strain was grown in enriched mineral salt medium (EMSM) using maltose 1 g/L, (NH4)2SO4 1 g/L as carbon and nitrogen source respectively, supplemented with 100 mg/L different dyes (pH 6.0), at 30 °C, 200 rpm from 48 to 96 h. The bacteria could aerobically decolorize dyes, such as Coomassie brilliant blue (95.42%), Bromcresol purple (93.34%), Congo red (72.37%) and Sarranine (61.7%), within 96 h. The dyes decolorization products were analyzed by ultra-violet and visible (UV-vis) spectroscopy before and after decolorization, which indicated that the four dyes were significantly degraded by the strain. The results indicated that the bacteria Bacillus amyloliquefaciens W36 could be used in dye-polluted wastewater treatment.

Purification and characterization of extracellular laccase produced by Ceriporiopsis subvermispora and decolorization of triphenylmethane dyes.

Laccases of white-rot fungi provide a promising future as a tool to be used in the field of biodegradation of synthetic dyes with different chemical structures. The aim of this study was production, characterization, and application of laccases from the white-rot fungus Ceriporiopsis subvermispora ATCC 90467 for decolorization of triphenylmethane dyes that could remain persistent in wastewater. Laccase was purified from a C. subvermispora culture by a four-step method resulting high specific activity of 2,571 U g-1 , 88-fold higher than crude laccase. Purified laccase (molecular weight 45 kDa) had the optimum activity at pH 2.0 and the optimum temperature 50 °C using ABTS as chromogenic substrate. Laccases efficiently decolorized triphenylmethane dyes such as Malachite Green (87.8%), Bromocresol Purple (71.6%), and Methyl Violet (68.1%) without redox mediator. However, decolorization percentage of hardly degradable triphenylmethane dyes such as Phenol Red, Bromophenol Blue, and Brilliant Blue R-250 was increased the presence of some low-molecular weight compounds (natural or synthetic redox mediators). Purified laccases were resistant to Mg2+ , Ca2+ , Ba2+ , Mn2+ , Fe2+ , Cu2+ , Zn2+ , and Sn2+ (10 mmol L-1 ). These findings suggest that laccases from C. subvermispora are able to decolorize triphenylmethane dyes without the negative influence of metal ions that can be found in wastewater.

Adsorption and transport of charged vs. neutral hydrophobic molecules at the membrane of murine erythroleukemia (MEL) cells.

The adsorption and transport of hydrophobic molecules at the membrane surface of pre- and post-DMSO induced differentiated murine erythroleukemia (MEL) cells were examined by time- and wavelength-resolved second harmonic light scattering. Two medium (<600 Da) hydrophobic molecules, cationic malachite green (MG) and neutral bromocresol purple (BCP), were investigated. While it was observed that the MG cation adsorbs onto the surface of the MEL cell, neutral BCP does not. It is suggested that an electrostatic interaction between the opposite charges of the cation and the MEL cell surface is the primary driving force for adsorption. Comparisons of adsorption density and free energy, measured at different pH and cell morphology, indicate that the interaction is predominantly through sialic acid carboxyl groups. MG cation adsorption densities have been determined as (0.6±0.3)×10(6) µm(-2) on the surface of undifferentiated MEL cells, and (1.8±0.5)×10(7) µm(-2) on differentiated MEL cells, while the deduced adsorption free energies are effectively identical (ca. -10.9±0.1 and -10.8±0.1 kcal mol(-1), respectively). The measured MG densities indicate that the total number of surface carboxyl groups is largely conserved following differentiation, and therefore the density of carboxylic groups is much larger on the differentiated cell surface than the undifferentiated one. Finally, in contrast to synthetic liposomes and bacterial membranes, surface adsorbed MG cations are unable to traverse the MEL cell membrane.

Identification of two bromocresol purple binding sites on human serum albumin.

BACKGROUND: In clinical settings, human serum albumin (HSA) is widely quantitated on the basis of change in the color of bromocresol purple (BCP) bound to HSA. However, the binding sites of BCP on HSA are hitherto unknown. We have identified these sites by performing binding experiments in which BCP was bound to HSA complexed with warfarin, ibuprofen, propofol, or myristic acid. METHODS: The binding experiment was performed with HSA complexed with a drug in buffers at pH 4.8 and 7.2. The number of BCP binding sites (n) and the association constant (Ka) were obtained by Scatchard analysis using the absorbance data of the BCP-HSA complexes. RESULTS: Two BCP binding sites on native HSA were identified at pH 7.2 and 1.3 sites were identified at pH 4.8. The number of sites for warfarin-HSA binding was approximately half that of native HSA at both pH values. The site numbers for myristate- and ibuprofen-HSA were 0.3 and 0.8 at pH 7.2, and 1.5 and 2.0 at pH 4.8, respectively; their Ka values were also high (18 x 10(4) and 10 x 10(4)). The Ka values for all HSA samples, except for the 2 samples, arranged from 1.5 x 10(4) to 3.3 x 10(4). The n and Ka values for propofol were equivalent to those for native HSA. CONCLUSION: We postulate that the 2 BCP binding sites include a warfarin-binding site (drug-binding site 1) and 1 of the 2 ibuprofen-binding sites.

Energetics and kinetics of photosynthetic water oxidation studied by photothermal beam deflection (PBD) experiments.

Determination of thermodynamic parameters of water oxidation at the photosystem II (PSII) manganese complex is a major challenge. Photothermal beam deflection (PBD) spectroscopy determines enthalpy changes (ΔH) and apparent volume changes which are coupled with electron transfer in the S-state cycle (Krivanek R, Dau H, Haumann M (2008) Biophys J 94: 1890­1903). Recent PBD results on formation of the Q⁻(A)/Y(•+)(Z) radical pair suggest a value of ΔH similar to the free energy change, ΔG, of -540±40 meV previously determined by the analysis of recombination fluorescence, but presently the uncertainty range of ΔH values determined by PBD is still high (±250 meV). In the oxygen-evolving transition, S3−−>S0, the enthalpy change may be close to zero. A prominent non-thermal signal is associated with both Q⁻(A)/Y(•+)(Z) formation (<1 µs) and the S3−−>S0 transition (~1 ms). The observed (apparent) volume expansion (ΔV of about +40 ų per PSII unit) in the S3−−>S0 transition seems to revert, at least partially, the contractions on lower S-transitions and may also comprise contributions from O2 and proton release. The observed volume changes show that the S3−−>S0 transition is accompanied by significant nuclear movements, which likely are of importance with respect to energetics and mechanism of photosynthetic water oxidation. Detailed PBD studies on all S-transitions will contribute to the progress in PSII research by providing insights not accessible by other spectroscopic methods.

Applications of a microplate reader in yeast physiology research.

Microplate readers have been useful assistants of researchers for several decades. This work is focused on the applications of a simple absorbance microplate reader in yeast physiology research, and its advantages and limitations in comparison with alternative methods are discussed. The two main procedures involved are measuring growth curves and monitoring the pH changes of medium using two different pH indicators. We suggest mathematical formulas for converting absorbance data into pH values. With a microplate reader as many as 96 samples can be simultaneously analyzed, while medium consumption is minimized to 100 microL per sample. The results can be observed in 24-48 h (for growth curves) or in 1-3 h (for pH changes) with minimal hands-on time required.

ATP leakage from yeast cells treated by extracellular glycolipids of Pseudozyma fusiformata.

The ustilaginaceous yeast Pseudozyma fusiformata secreted glycolipids which were lethal to many yeasts and fungi more active at pH of about 4.0, and in the temperature range of 20-30 degrees C. Purified glycolipids enhanced non-specific permeability of the cytoplasmic membrane in sensitive cells, which resulted in ATP leakage and susceptibility of the cells to staining with bromocresol purple. Cells of Saccharomyces cerevisiae lost the ability to acidify the medium. Basidiomycetous yeasts were more sensitive to the glycolipids than ascomycetous ones. The minimal effective glycolipid concentration was 0.13 and 0.26 mg ml(-1) for Cryptococcus terreus and Filobasidiella neoformans, while for Candida albicans and Saccharomyces cerevisiae it was 1.0 and 1.6 mg ml(-1).

Zymographic detection of cinnamic acid decarboxylase activity.

The manuscript includes a concise description of a new, fast and simple method for detection of cinnamic acid decarboxylase activity. The method is based on a color shift caused a by pH change and may be an excellent procedure for large screenings of samples from natural sources, as it involves no complex sample processing or purification. The method developed can be used in preliminary approaches to biotransformation processes involving detection of hydroxycinnamic acid decarboxylase activity.

Kinetics of proton uptake and dye binding by photoactive yellow protein in wild type and in the E46Q and E46A mutants.

We studied the kinetics of proton uptake and release by photoactive yellow protein (PYP) from Ectothiorhodospira halophila in wild type and the E46Q and E46A mutants by transient absorption spectroscopy with the pH-indicator dyes bromocresol purple or cresol red in unbuffered solution. In parallel, we investigated the kinetics of chromophore protonation as monitored by the rise and decay of the blue-shifted state I(2) (lambda(max) = 355 nm). For wild type the proton uptake kinetics is synchronized with the fast phase of I(2) formation (tau = 500 micros at pH 6.2). The transient absorption signal from the dye also contains a slower component which is not due to dye deprotonation but is caused by dye binding to a hydrophobic patch that is transiently exposed in the structurally changed and partially unfolded I(2) intermediate. This conclusion is based on the wavelength, pH, and concentration dependence of the dye signal and on dye measurements in the presence of buffer. SVD analysis, moreover, indicates the presence of two components in the dye signal: protonation and dye binding. The dye binding has a rise time of about 4 ms and is coupled kinetically with a transition between two I(2) intermediates. In the mutant E46Q, which lacks the putative internal proton donor E46, the formation of I(2) is accelerated, but the proton uptake kinetics remains kinetically coupled to the fast phase of I(2) formation (tau = 100 micros at pH 6.3). For this mutant the protein conformational change, as monitored by the dye binding, occurs with about the same time constant as in wild type but with reduced amplitude. In the alkaline form of the mutant E46A the formation of the I(2)-like intermediate is even faster as is the proton uptake (tau = 20 micros at pH 8.3). No dye binding occurred in E46A, suggesting the absence of a conformational change. In all of the systems proton release is synchronized with the decay of I(2). Our results support mechanisms in which the chromophore of PYP is protonated directly from the external medium rather than by the internal donor E46.

Correlation of the suicide phenomenon in Aeromonas species with virulence and enteropathogenicity.

Certain strains of mesophilic Aeromonads like A. hydrophila, A. veronii biotype sobria and A. caviae when grown in broth containing 0.5% glucose, undergo growth inhibition concomitant with acetate accumulation. As these strains become nonviable after 24 h, this phenomenon is termed suicide. We investigated suicidal strains of Aeromonas species as means of understanding animal virulence and enteropathogenicity. Non suicidal strains of A. Hydrophila showed and overall 88.8% lethality rate and non suicidal strains of A. veronii biotype sobria showed 83.3% lethality rate and was nil for its suicidal part. Of the two suicidal A. caviae strains tested, none were lethal. The present data suggest that the suicide phenomenon may explain strain specific [A. veronii biotype sobria, A. hydrophila] and species specific [A. caviae] virulence and enteropathogenicity.

Proton transfer reactions linked to rhodopsin activation.

Purified bovine rhodopsin solubilized in dodecyl maltoside was photolyzed at 20 degreesC with 477 nm light, and difference spectra were collected at time delays ranging from 10 micros to 10 ms after photolysis. Bromocresol purple was added to the samples to detect pH changes in the aqueous environment due to changes in the protonation state of rhodopsin. The data were analyzed using singular value decomposition and global exponential fitting, which revealed three exponential processes indicating the presence of at least four intermediates. Spectral changes of the indicator dye were separated from those of rhodopsin, and proton release and uptake rates were analyzed within the framework of rhodopsin photoreaction kinetics. Proton release occurred during Lumi decay to Meta-I380 followed by uptake upon Meta-I380 decay and by a more significant proton uptake with the time course of Meta-I480 decay. On the basis of the estimated number of protons released and taken up in each step of the rhodopsin photoreaction, we concluded that two forms of Meta-II are present. The two forms of Meta-II, Meta-IIa' and Meta-IIb, differ in protonation state from one another as do both from the earlier, 380 nm absorbing form, Meta-I380.

Multivariate analysis of the association of bromocresol purple anion with bovine serum albumin.

The interaction of bromocresol purple (BCP) anions with bovine serum albumin (BSA) was investigated by principal factor analysis method, and reaction model, the number of molecular species and spectra of each component present in the reaction mixture were determined. The number of molecular species concerning the absorption intensity was three, including free BCP anion. Most part of the spectral change could be explained by the monomer binding of bromocresol purple anion (D) to serum albumin (P), a simple one step equilibrium, P + D = PD. A second type of association of BCP anions with serum albumin was also present, though in a small amount. Of six models tested which consisted of three or four molecular species, the sequential two step reaction model, P = PD = PD2, was the best model to explain the spectral data, and an existence of BCP anions as a dimer on the serum albumin was demonstrated. The dissociation constants were estimated at K1 = 1.6 x 10(-6) M for the first step and K2 = 1.2 x 10(-5) M for the second step.

Evaluation of a broth microdilution antifungal susceptibility test with a pH indicator: comparison with the broth macrodilution procedures.

A broth microdilution antifungal susceptibility test with a pH indicator (bromocresol purple) in a synthetic medium was evaluated. This method measures cellular activity instead of simply a change in biomass. The variations of pH caused by fungal activity were measured by changes in optical densities at 450 nm. The MICs50 obtained were compared with the MICs found by the classical broth macrodilution procedure. In most cases broth micro- and macrodilution MICs were in agreement for amphotericin B, fluconazole, flucytosine and nystatin.

Specific association of bromocresol purple anions with a magnesium complex of a phosphorylated intermediate during steady-state hydrolysis of ATP by the Mg2+ + Ca2+-dependent ATPase of sarcoplasmic reticulum.

The mechanism of dimeric binding of bromocresol purple (BCP) anions to Mg2+ + Ca2+-ATPase of the sarcoplasmic reticulum (SR) and the resulting partial inhibition of the ATPase activity were studied. BCP anions in three states, free monomer, bound monomer, and bound dimer, were spectrophotometrically calculated by solving simultaneous equations, delta A lambda 1-lambda 2 = sigma delta ai (epsilon i lambda 1-epsilon i lambda 2), and concentration changes of these states were analyzed. The addition of ATP caused an increase in the bound dimer and a decrease in the free monomer, but the change of the bound monomer was slight. The decrease in delta A (decrease phase) on the addition of ATP on dual-wavelength spectrophotometry at 585-610 nm was related to an increase in the amount of dimer bound to the SR membranes. The magnitude of the decrease phase increased with an increase in Mg2+ concentration and decreased with an increase in the concentration of Ca2+. BCP anions at the probe concentration partially inhibited the ATPase activity, and brought about a decrease in the ADP-sensitive E-P (E1P) and an increase in the ADP-insensitive E-P (E2P), though BCP anions did not affect the amount of total E-P. On elimination of Mg2+ at the steady-state E-P level both E2P and E2P . (BCP)2 were decomposed, suggesting that the enzyme form binding the BCP dimer was Mg . E-P. An increase in Mg2+ concentration increased E2P but an increase in Ca2+ concentration decreased E2P. Decomposition of E2P to P1 was inhibited by BCP anions. The following simple scheme was suggested to explain the partial inhibition of the ATPase activity, (Formula: see text). Application of BCP anions was discussed for use as a probe for Mg . E-P in the steady-state ATP hydrolysis.

Temperature-jump-induced release of hydrogen ions from chloroplasts and its relaxation characteristics in the presence of ionophores.

Temperature-jump-induced absorption changes of a bromocresol purple in chloroplast suspensions in the dark were studied. After a rapid rise in temperature (less than 10 mus), a slow absorbance decrease of bromocresol purple (t1/2 approximately 0.2 s) following a fast absorbance decrease of chloroplasts and bromocresol purple (t1/2 less than 1 ms) was observed. The slow absorbance decrease corresponds to acidification of the suspending medium, indicating H+ efflux from chloroplasts after the temperature jump. Nigericin and gramicidin D suppressed the slow absorbance change completely in the presence of 10 mM KC1, while valinomycin did not affect it. The fast absorbance change was not affected by the above ionophores. 3-(3,4-dichlorophenyl)-1,1-dimethylurea also diminished the slow absorbance change.